Li Dan, Hong Jie, Cao Weibiao
Departments of Medicine (D.L., W.C.) and Pathology (W.C.), Rhode Island Hospital and Warren Alpert Medical School of Brown University, Providence, Rhode Island; and Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine, Renji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China (J.H.).
Departments of Medicine (D.L., W.C.) and Pathology (W.C.), Rhode Island Hospital and Warren Alpert Medical School of Brown University, Providence, Rhode Island; and Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine, Renji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China (J.H.)
J Pharmacol Exp Ther. 2017 Jan;360(1):14-22. doi: 10.1124/jpet.116.236620. Epub 2016 Oct 18.
We have shown that NADPH oxidase (NOX)5-S may mediate the acid-induced decrease in cell apoptosis. However, mechanisms of NOX5-S-dependent decrease in cell apoptosis are not fully understood. In this study, we found that silencer-of-death domain (SODD) was significantly increased in esophageal adenocarcinoma (EA) tissues, EA cell lines FLO and OE33, and a dysplastic cell line CP-B. Strong SODD immunostaining was significantly higher in low-grade dysplasia (66.7%), high-grade dysplasia (81.2%), and EA (71.2%) than in Barrett's mucosa (10.5%). Acid treatment significantly increased SODD protein and mRNA expression and promoter activity in FLO cells, an increase that was significantly decreased by the knockdown of NOX5-S and nuclear factor κB (NF-κB)1 p50 with their small interfering RNAs. Similarly, acid-induced increase of SODD mRNA was blocked by knockdown of NOX5-S and p50 in a BE cell line CP-A. Overexpression of NOX5-S significantly increased SODD protein expression in FLO cells. Moreover, overexpression of NOX5-S or p50 significantly increased the SODD promoter activity and decreased the caspase 9 activity or apoptosis. NOX5-S overexpression-induced increase in SODD promoter activity was significantly decreased by knockdown of p50. In addition, acid treatment significantly decreased the caspase 9 activity, a decrease that was significantly inhibited by knockdown of SODD. Furthermore, chromatin immunoprecipitation assay showed that NF-κB1 p50 bound to SODD genomic DNA containing a NF-κB-binding element GGGGACACCCT. This binding element was further confirmed by a gel mobility shift assay. We conclude that acid-induced increase in SODD expression and decrease in cell apoptosis may depend on the activation of NOX5-S and NF-κB1 p50 in FLO cells.
我们已经表明，NADPH氧化酶（NOX）5-S可能介导酸诱导的细胞凋亡减少。然而，NOX5-S依赖性细胞凋亡减少的机制尚未完全了解。在本研究中，我们发现死亡结构域沉默蛋白（SODD）在食管腺癌（EA）组织、EA细胞系FLO和OE33以及发育异常细胞系CP-B中显著增加。低级别发育异常（66.7%）、高级别发育异常（81.2%）和EA（71.2%）中强烈的SODD免疫染色显著高于巴雷特黏膜（10.5%）。酸处理显著增加了FLO细胞中SODD蛋白和mRNA表达以及启动子活性，用小干扰RNA敲低NOX5-S和核因子κB（NF-κB）1 p50可显著降低这种增加。同样，在BE细胞系CP-A中，敲低NOX5-S和p50可阻断酸诱导的SODD mRNA增加。NOX5-S的过表达显著增加了FLO细胞中SODD蛋白表达。此外，NOX5-S或p50的过表达显著增加了SODD启动子活性并降低了半胱天冬酶9活性或细胞凋亡。敲低p50可显著降低NOX5-S过表达诱导的SODD启动子活性增加。此外，酸处理显著降低了半胱天冬酶9活性，敲低SODD可显著抑制这种降低。此外，染色质免疫沉淀试验表明，NF-κB1 p50与含有NF-κB结合元件GGGGACACCCT的SODD基因组DNA结合。凝胶迁移率变动分析进一步证实了这种结合元件。我们得出结论，酸诱导的SODD表达增加和细胞凋亡减少可能取决于FLO细胞中NOX5-S和NF-κB1 p50的激活。
